Immunoprotective recombinant antigen from anaplasma marginale, vaccine compositions and methods of use

ABSTRACT

Vaccines and methods useful to induce an immune response which is protective to reduce the severity or prevent infection by ehrlichial parasites of the species  Anaplasma marginale  utilizing recombinant MSP1a surface protein antigens alone or in combination with tick cell culture derived  A. marginale.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of prior filed, copendingU.S. provisional patent application Serial No. 60/244,333, filed Oct.30, 2000, which application is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field

[0003] The present invention relates to antigenic polypeptides andproteins, related vaccines and methods useful to induce an immuneresponse which is protective to reduce the severity or prevent infectionby ehrlichial parasites of the species Anaplasma marginale.

[0004] 2. Background

[0005] Anaplasmosis is a tick-borne disease of cattle caused by theobligate intraerythrocytic ehrlichial pathogen A. marginale. The acutephase of the disease is characterized by severe anemia, weight loss,fever, abortion, lower milk production and often death. The only knownsite of development of A. marginale in cattle is within bovineerythrocytes. The number of infected erythrocytes increaseslogarithmically and removal of these infected cells by phagocytosisresults in development of anemia and icterus without hemoglobinemia andhemogloinuria. Biological transmission of A. marginale is effected byfeeding ticks, while mechanical transmission occurs when infected bloodis transferred to susceptible animals by biting flies or byblood-contaminated fomites. Cattle that recover from acute infectionremain persistently infected and serve as reservoirs for mechanicaltransmission and infection of ticks. Approximately 20 species of tickshave been incriminated as vectors worldwide. The developmental cycle ofA. marginale in ticks is complex and coordinated with the tick feedingcycle. After infection and development of A. marginale in tick gutcells, many other tick tissues become infected, including the salivaryglands from where the ehrlichia is transmitted to vertebrates duringfeeding.

[0006] MSP1 is one of six major surface proteins (MSPs) that have beendescribed on A. marginale from bovine erythrocytes and has been found tobe conserved on tick salivary gland-derived A. marginale [1]. MSP1 is aheterodimer composed of two structurally unrelated polypeptides: MSP1awhich is encoded by a single gene, msp1α, and MSP1b which is encoded byat least two genes, msp1β1 and msp1β2 [2, 3]. MSP1a is variable inmolecular weight among geographic isolates because of a variable numberof tandem 28 or 29 amino acid repeats in the amino terminal of theprotein. Immunization of cattle with affinity-purified native MSP1complex has previously been shown to induce protective immunity incattle that received homologous or heterologous challenge with A.marginale geographic isolates [4, 5]. In addition, MSP1a and MSP1bexpressed by recombinant Escherichia coli were shown to be putativeadhesins to bovine erythrocytes [6, 7]. Although the MSP1 complex hasbeen suggested to be involved in erythrocyte invasion, its role ininfection and multiplication of the parasite in the tick vector has notbeen reported.

[0007] Recently, A. marginale has been grown in continuous culture in acell line, IDE8, derived from embryos of the tick Ixodes scapularis [8].See also U.S. Pat. No. 5,869,335, incorporated herein by reference. TheVirginia isolate of A. marginale was initially propagated in the IDE8tick cell line but subsequently an Oklahoma isolate was propagated inthe tick cell line and characterized [9]. Colonies of A. marginale incultured tick cells were morphologically similar to those observed inticks [8-10], and A. marginale harvested from cell culture wereinfective for both cattle and ticks. All 6 MSPs of A. marginale werefound to be conserved on the cell culture-derived organisms and theantigenic composition remained the same after successive passage in cellculture. The A. marginale isolate antigenic identity, as determined bythe molecular weight of the MSP1a, was retained in culture [9, 11].

[0008] MSP1 complex isolated A. marginale also has been utilized for thevaccination of ruminants, as disclosed by McGuire et al. in U.S. Pat.No. 5,549,898 issued Aug. 27, 1996 (said patent being incorporatedherein by reference).

[0009] Existing vaccines, however, including formulations usingpartially purified parasites from infected erythrocytes and culturedtick cells, are currently handicapped by mechanisms developed by theparasite to hide the most relevant epitopes from the host immune system.The inclusion of recombinant protein preparations in vaccineformulations would allow the development of host immune response againstrelevant epitopes not available for the host immune system in naturalconformations present in whole-parasite or parasite-derived purifiedantigens.

SUMMARY OF THE INVENTION

[0010] Experimental results described by the inventors in copending U.S.provisional patent application Serial No. 60/244,333 demonstrate adifferential role for MSP1a and MSP1b polypeptides of the MSP1 surfaceprotein complex for adherence of A. marginale to bovine erythrocytes andtick cells. Recombinant MSP1a expressed in E. coli was shown to be anadhesin for bovine erythrocytes and both native and cultured tick cells.In contrast, recombinant E. coli expressing MSP1b adhered only to bovineerythrocytes and not to tick cells. The role of the MSP1 complex,therefore, was determined to vary among vertebrate and invertebratehosts.

[0011] The present invention is based upon the surprising discovery thatcattle immunized with the recombinant MSP1a surface protein antigen ofthe MSP1 complex alone or in combination with tick cell culture-derivedA. marginale are better protected against A. marginale infection asdemonstrated by a lower reduction in packed cell volume (PCV) and lowerpeak parasitemia (PPE) than cattle immunized with the MSP1 complex, acombination of uncomplexed MSP1a and MSP1b surface protein antigens, theMSP1b antigen alone, cell culture derived A. marginale, or cell culturederived A. marginale combined with MSP1b. Indeed, onlyerythrocyte-derived A. marginale appears to confer like protection.

[0012] Thus, in one embodiment of the present invention there isprovided a vaccine composition for inducing an immune response in aruminant, the vaccine composition comprising the recombinant MSP1apurified surface protein antigen of A. marginale or subunits thereof,alone or in combination with other antigenic components, wherein thevaccine composition further comprises a pharmaceutically acceptablecarrier or diluent.

[0013] In another embodiment of the present invention there is provided,a method for inducing an immune response in a ruminant to provide immuneprotection which reduces the severity of or prevents infection by A.marginale, the method comprising administering to said ruminant aneffective amount of the inventive vaccine composition.

[0014] A better understanding of the present invention, its severalaspects, and its advantages will become apparent to those skilled in theart from the following detailed description, taken in conjunction withthe attached figures, wherein there is described the preferredembodiment of the invention, simply by way of illustration of the bestmode contemplated for carrying out the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a graphical illustration of the immune response againstMSP1a and MSP1b determined by Western blot analysis of sera derived fromimmunized cattle and controls generated in connection with theexperimental results reported herein.

[0016]FIG. 2 is a graphical illustration of the reduction in PCVachieved by various combinations of antigens and controls in connectionwith the experimental results reported herein.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Before explaining the present invention in detail, it isimportant to understand that the invention is not limited in itsapplication to the details of the embodiments and steps describedherein. The invention is capable of other embodiments and of beingpracticed or carried out in a variety of ways. It is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and not of limitation.

[0018] In accordance with the present invention there is provided a newvaccine against the ehrlichial cattle pathogen A. marginale through theuse of discrete recombinant MSP1a and polypeptides derived from MSP1acontaining the immunoprotective and functional regions that areexpressed in E.coli. In one aspect, only recombinant MSP1a orimmunoprotective and functional regions thereof are utilized as theantigenic component of the vaccine. In another aspect, recombinant MSP1aor subunits thereof are utilized in combination with other antigenpreparations, particularly antigen preparations derived from A.marginale-infected cultured tick IDE8 cells.

[0019] MSP1a and MSP1b are isolated from A. marginale initial bodies asa complex of two noncovalently linked, antigenically distinctpolypeptides. It is possible that the association between MSP1a andMSP1b in the surface protein complex allows the parasite to moreeffectively bind to erythrocyte and/or tick cell components. MSP1a couldbe the essential subunit in the recognition of the tick cell receptor,while the binding to the erythrocyte receptor could be mediatedprimarily by MSP1b or by both protein subunits through the binding ofdistinct erythrocyte components. Additionally, the association betweenMSP1a and MSP1b could stabilize and/or properly conform the MSP1 complex[6].

[0020] MSP1a is encoded by a single monocystronic gene, msp1α, which ispolymorphic among geographical isolates of A. marginale [12, 13, 15]. A.marginale isolates differ in the number of 28-29 amino acids tandemrepeats within the MSP1a polypeptide [13, 15], which contain aneutralization-sensitive epitope [4, 13]. However, the sequence of msp1αa does not change during the multiplication of the parasite in thebovine host and the tick vector. The second MSP1 subunit, MSP1b, isencoded by at least two monocystronic genes, msp1β1 and msp1β2 [3].These loci are polymorphic between and within populations of A.marginale from different geographical regions and life cycle stages butconserve a high degree of similarity. Sequence diversity is mainly dueto point mutations in variable regions, perhaps due to selective immunepressure. The genetic structure of msp1 α and β genes together with thevital function of codified polypeptides permits the inclusion ofrecombinant MSP1 polypeptides, or its functional domains, in vaccineformulations against A. marginale.

[0021] The experiments described and examples provided hereinafterdemonstrate that cattle immunized with recombinant MSP1a alone or incombination with tick cell culture derived A. marginale are unexpectedlybetter protected against A. marginale infection as demonstrated by alower reduction in packed cell volume (PCV) and lower peak parasitemia(PPE) than cattle immunized with the MSP1 complex, a combination ofuncomplexed MSP1a and MSP1b surface protein antigens, the MSP1b antigenalone, cell culture derived A. marginale, or cell culture derived A.marginale combined with MSP1b. Indeed, only erythrocyte-derived A.marginale appears to confer like protection.

EXAMPLE 1 Preparation of Recombinant E. coli Expressing MSP1 α andPreparation of Antigen

[0022] The msp1a gene was cloned by PCR from the Oklahoma isolate of A.marginale derived from infected erythrocytes. DNA was extracted from 1ml stored blood samples containing infected bovine erythrocytescollected during high parasitemia employing 250 μL Tri Reagent (Sigma)and following manufacturer's recommendations. Extracted DNA wasresuspended in 100 μL water. The msp1a gene was amplified from 1 μL DNAby PCR using 10 pmol of each primer MSP1aP: 5′GCATTACAACGCAACGCTTGAG3′and MSP1a3: 5′GCTTTACGCCGCCGCCTGCGCC3′ in a 50-μL volume PCR employingthe Access RT-PCR system (Promega). Reactions were performed in anautomated DNA thermal cycler (Eppendorf) for 35 cycles. After an initialdenaturation step of 30 sec at 94° C., each cycle consisted of adenaturing step of 30 sec at 94° C. and an annealing-extension step of2.5 min at 68° C. The program ended by storing the reactions at 4° C.PCR products were electrophoresed on 1% agarose gels to check the sizeof amplified fragments. The amplified fragments were resin purified fromPCR reactions (Wizard Promega) and cloned into pGEM-T vector (Promega)for sequencing both strands (Core Sequencing Facility, Department ofBiochemistry and Molecular Biology, Noble Research Center, OklahomaState University).

[0023] For high level expression of MSP1a, msp1 α coding region wasamplified from per1 (msp1 α in pGEM-T vector) plasmid DNA by PCR usingthe primers 5′CCGCTCGAGATGTTAGCGGAGTATGTGTCC3′ and5′GAAGATCTCGCCGCCGCCTGCGCC3′. The msp1 α amplification product wasdigested with XhoI and BglII and inserted into the cloning site ofpFLAG-CTC expression vector (Sigma). Recombinant plasmid was namedpFLC1a. In this construct, the inserted gene is under the control of theinducible tac promoter and yield full-length MSP1a polypeptide, with aC-terminal fusion of a FLAG marker octapeptide. The fidelity andorientation of the construct was verified by sequencing. For expressionof MSP1a recombinant polypeptides, pFLC1a expression plasmid wastransformed into E. coli K-12 (strain JM109). Transformed E. colistrains were inoculated in LB containing 50 μ/ml Ampicillin and 0.4%glucose. Cultures were grown at 37° C. to OD_(600nm)=0.4. IPTG was thenadded to 0.5 mM final concentration, and incubation continued during 4h, for induction MSP1a expression. Cells were collected bycentrifugation and membranes extracted after sonication andcentrifugation. MSP1b was cloned, expressed and purified in a similarway. Doses of 5 ml containing 100 μg recombinant antigens were used forvaccination in subsequent studies.

EXAMPLE 2 Analysis of the Protective Capacity of Vaccine PreparationsContaining Recombinant MSP1a

[0024] 1 Propagation of Anaplasma marginale in Tick Cell Culture andPreparation of Immunogen.

[0025] The IDE8 (ATCC CRL 11973) tick cell line derived from embryos ofIxodes scapularis was maintained at 31° C. in L-15B medium, pH 7.2,supplemented with 5% heat inactivated fetal bovine serum (FBS; Sigma,USA), 10% tryptose phosphate broth (Difco, USA) and 0.1% bovinelipoprotein concentrate (ICN, USA). Cultures were grown in 25-cm²plastic flasks (Nunc, Rosekilde, Denmark) with 5 ml of medium, and themedium was replaced weekly. The cells were subcultured at 1:5 to 1:20,and the cells became tightly adherent to the culture substrate andmultiplied with a population doubling time of 3 to 5 days to a densityof about 5×10⁶ cells/ml. Nearly confluent monolayers from each passagewere collected and stored in liquid nitrogen in medium with 10% DMSO.

[0026] Tick cell cultures infected with the Oklahoma isolate of A.marginale were propagated. Terminal cell cultures were harvested, thecells centrifuged, and the contents of each T25 flask was resuspended in1 ml PBS and stored at −70° C. until used as antigen for immunogendoses. The antigen aliquots were thawed, pooled and a sample was takenand tested by indirect ELISA. The cell culture-derived antigen wasinactivated with beta propiolactone (BPL) and the volume was adjusted to5 ml so that each dose contained approximately 2×10¹⁰ A. marginale.

[0027] 2. Preparation of A. marginale Antigen from Bovine Erythrocytes.

[0028] Two susceptible, splenectomized calves (PA432 and PA433) wereeach inoculated with 2.5 ml blood stabilate (40% parasitemia) collectedfrom a calf with the Virginia isolate of A. marginale. The calves weremonitored for infection by examination of stained blood smears. Bloodwas collected from PA432 at parasitemias of 13.6% and 32.7% and fromPA433 at parasitemias of 12.2% and 12.9%. After each collection, theerythrocytes were washed 3 times in PBS, each time removing the buffycoat. The erythrocytes were frozen at −70° C. 1:1 in RPMI cell culturemedium until used as antigen for the immunization studies. The frozenerythrocyte antigen was thawed, washed in PBS, and centrifuged. Theresulting pellet was washed to remove the hemoglobin, after which theantigen was pooled and inactivated with BPL. An aliquot was tested byELISA as described previously for the erythrocyte antigen preparationusing a known erythrocyte standard. Doses (5 ml) were prepared thatcontained approximately 2×10¹⁰ A. marginale.

[0029] 3. Experimental Design.

[0030] Fifty, 16-month month old Angus cattle were randomly assignedinto ten groups of five cattle each that were immunized with variousantigens as follows. (1) MSP1 complex, (2) MSP1a and MSP1b, (3) MSP1a,(4) MSP1b, (5) cell culture-derived A. marginale, (6) cellculture-derived A. marginale and MSP1a, (7) cell culture-derived A.marginale and MSP1b, (8) erythrocyte-derived A. marginale, (9)uninfected IDE8 tick cells and (10) adjuvant only.

[0031] 4. Immunizations.

[0032] All cattle were immunized 3 times by subcutaneous injection ofthe antigen at weeks 1, 4 and 6. Each antigen dose was 5 ml in volumeand contained an antigen in the adjuvant, Xtend® III (GrandLaboratories, Larchwood, Iowa). All cattle were challenge-exposed 10weeks after the last immunization with 1×10⁷ A. marginale infectederythrocytes collected from a calf experimentally infected with theOklahoma isolate of A. marginale. Blood of the immunized and controlcattle was monitored for infection with A. marginale by microscopicexamination of blood smears and hematology was done daily after theonset of infection. Parameters evaluated in cattle includeddetermination of the peak percent infected erythrocytes (PPE), percentreduction in the packed cell volume (PCV), and the prepatent period(days) determined from the day of challenge-exposure to the onset ofinfection.

[0033] 5. Collection of Blood and Serum Samples.

[0034] Whole blood was collected in vacutainer tubes containing EDTA andused for preparation of stained blood smears for light microscopy andfor determination of the PCV. Serum samples were collected from eachanimal before immunization, weekly until the cattle werechallenge-exposed and daily after cattle developed parasitemia as aresult of challenge-exposure. Serum samples were stored at −70° C. untiltested by competitive ELISA and Western blots.

[0035] 6. Characterization of the Immune Response in Vaccinated Cattleby Competitive ELISA and Western Blots.

[0036] Antibody responses of all immunized and control cattle at twoweeks after the last immunization to MSP1a, MSP1b and MSP5 weredetermined using ELISAs specific for detection of antibodies to each ofthese MSPs. Antibody responses of all immunized and control cattle attwo weeks after the last immunization to MSP1a and MSP1b were alsoanalyzed by Western blot. One hundred micrograms of recombinant MSP1a orMSP1b were loaded in an 8% polyacrylamide gel. SDS-PAGE gels weretransferred to a nitrocellulose membrane. The membrane was blocked with5% skim milk for 1 hr at room temperature. Sera from immunized cattlewas diluted 1:200 in TBS. Serum from an uninfected bovine was includedas a negative control. All sera were incubated with the membrane for 1hr at room temperature using a Mini-Protean II Multi-screen (BioRad,USA). The membrane was washed 3 times with TBST and incubated for 1 hrat room temperature with goat anti-rabbit IgG alkaline phosphataseconjugate (KPL, USA) diluted 1:10,000. The membrane was washed again andthe color developed using Sigma Fast BCIP/NBT alkaline phosphatasesubstrate tablets. The membrane was then examined for recognition of thebands corresponding to MSP1a and MSP1b.

[0037] 7. Statistical Analysis.

[0038] For the analysis of results from the immunization experiment,pairwise comparisons (Student's t test) were conducted to compareresults between cattle immunized with antigen preparations and thecontrols. Parameters analyzed included the prepatent period (days), thepeak percent parasitized erythrocytes (PPE) and the percent reduction inthe packed cell volume (PCV). Mean antibody levels were compared usingan ANOVA test.

[0039] 8. Results.

[0040] Antibody titers against MSP1a, MSP1b and MSP5 in immunized cattlepeaked two weeks after the last immunization. The immune responseagainst MSP1a, MSP1b and MSP5 was analyzed by Western blot. Cattleimmunized with recombinant antigen preparations responded to recombinantproteins included on each preparation (FIG. 1). Cattle immunized withtick cell derived A. marginale antigens and with infectederythrocytes-derived antigens recognized primarily MSP1b or MSP1a,respectively (FIG. 1).

[0041] Protection was evaluated using the reduction in PCV, the PPE andthe prepatent period. No differences were observed in the prepatentperiod. The PPE was reduced in cattle immunized with MSP1a, MSP1b, thecombination of recombinant antigens with infected tick cells-derivedantigens and in animals immunized with infected erythrocytes-derivedantigens as shown in Table 1. TABLE 1 Peak Parasitemia (%) Group Ave SDP MSP1 5.5 2.8 0.13 1a + 1b 6.0 1.6 0.14 1a 4.8 0.6 0.03 1b 3.9 1.0 0.01TC 4.1 2.3 0.03 1a + TC 4.7 1.4 0.03 1b + TC 3.9 0.8 0.01 RBC 2.7 1.1 0.004 Saline 5.5 1.4 0.08 Cells 7.4 2.3 —

[0042] The reduction in PCV, associated with clinical signs, wassignificantly reduced in cattle immunized with MSP1a combined withinfected tick cell-derived antigens and in cattle immunized witherythrocyte-derived antigens (See FIG. 2, wherein Reduction PCV=[(AveStart PCV−Lowest PCV)/Start PCV]×100).

[0043] The results of these experiments demonstrated that:

[0044] a. Cattle immunized with infected tick cell-derived antigens hada preferential recognition for MSP1b while cattle immunized witherythrocyte-derived antigens showed a bias toward MSP1a. The bias in theantibody response against MSP1a or MSP1b in cattle immunized with A.marginale antigens from IDE8 tick cells or bovine erythrocytes suggeststhat the MSP1 complex exposure on the surface of parasites may varyduring multiplication on the tick and mammalian hosts;

[0045] b. The immunization with the MSP1 complex or with MSP1a and MSP1btogether did not protect cattle after challenge with A. marginaledespite that cattle responded to both antigens; and

[0046] c. Cattle with a predominant immune response against MSP1a(groups immunized with MSP1a, MSP1a plus infected tick cell-derivedantigens and infected erythrocyte-derived antigens) were protectedagainst A. marginale infection as demonstrated by the lower reduction inPCV.

[0047] It can thus be appreciated that the utilization of recombinantMSP1a in vaccines provides an advantageous mechanism to achieveresistance in cattle against A. marginale infection. Whereaserythrocyte-derived A. marginale is disadvantaged due to cost,difficulties in purifying antigen from bovine membranes, problems withpreventing pathogen contamination and difficulties in standardization,recombinant MSP1a may be readily and cost effectively prepared in astandardized, pure form free of bovine erythrocyte membranes andantigens that might result in formation of an immune response to bovineblood cells.

EXAMPLE 3 Function of MSP1a Tandem Repeats in Adhesion to Host CellReceptors

[0048] 1. Construction, Expression in E. coli and Characterization ofWild Type MSP1a and Mutants.

[0049] A MSP1a (Oklahoma isolate msp1α clone per1 [14]) mutant lackingthe tandem repeats was constructed by PCR. Oligonucleotide primers RI0R(5′-CCGAATTCCATGTTAGCGGCTAATTGGCGGCAAGAGATGCG-3′) and MSP1a3BII(5′-CCAGATCTCTTTACGCCGCCGCCTGCGCC-3′) were designed to amplify the msp1αgene lacking 6 amino acids preceding the repeats and the tandem repeatsin a 50 μl volume PCR (0.2 μM each primer, 1.5 mM MgSO₄, 0.2 mM dNTP, 1XAMV/Tfl reaction buffer, 5u Tfl DNA polymerase) employing the AccessRT-PCR system (Promega, USA). Reactions were performed in an automatedDNA thermal cycler (Eppendorf Mastercycler® personal, USA) for 35cycles. After an initial denaturation step of 30 sec at 94° C., eachcycle consisted of a denaturing step of 30 sec at 94° C. and anannealing-extension step of 2.5 min at 68° C. The program ended bystoring the reactions at 4° C. The primers introduced an ATG initiationcodon and Eco RI and Bgl II restriction sites for cloning into thepFLAG-CTC expression vector (Sigma). The resulting plasmid pAF0R1 wastransformed into E. coli JM109 and induced for expression of mutantMSP1a as previously reported for MSP1a [14]. For the expression of MSP1a(Oklahoma isolate msp1α clone per1 [14]) tandem repeats in E. coli, thisregion was amplified using oligonucleotide primers RNOKBS5(5′-GAGATCTGCTGATGGCTCGTCAGCGGG-3′) and RNOKBS3(5′-GGTCGACCCTGATTGAGACGATGTACTGGCC-3′). The PCR was conducted aspreviously described but with amplification cycles consisting of adenaturing step of 30 sec at 94° C., an annealing step of 30 sec at 58°C. and an extension step of 1 min at 68° C. The 5′ and 3′ amplificationprimers contained Bgl II and Sal I restriction sites, respectively, forcloning into pFLC1b [14] for expression in E. coli as a fusion peptideto the COOH-terminus of MSP1b (locus β1, Oklahoma isolate). Theresulting plasmid pF1bRNO4 was transformed into E. coli JM109 andinduced for expression of mutant MSP1b>MSP1a-repeats protein aspreviously reported for MSP1b [14]. All constructs were sequenced at theCore Sequencing Facility, Department of Biochemistry and MolecularBiology, Noble Research Center, Oklahoma State University using ABIPrism dye terminator cycle sequencing protocols developed by AppliedBiosystems (Perkin-Elmer Corp., Foster City, Calif.).

[0050] Expression of recombinant mutant proteins was assayed bySDS-PAGE, Western blot or live-cell immunofluorescence assay aspreviously reported [14]. The hemagglutination of bovine erythrocytesand adhesion to cultured IDE8 tick cells of recombinant E. coliexpressing the wild type and mutant proteins was evaluated in amicrotitre hemagglutination and E. coli recovery adhesion assays,respectively, as reported [14].

[0051] 2. Results.

[0052] To study the function and structural organization of tandemrepeated peptides in MSP1a we selected a recent A. marginale fieldisolate from Oklahoma that is tick-transmissible and for which we havecloned and characterized MSP1a and MSP1b. A recombinant mutant MSP1a wasconstructed lacking the tandem repeated peptides and expressed in E.coli. The recombinant mutant protein was expressed at high levels andwas secreted to the E. coli membrane as shown by live-cellimmunofluorescence. Nevertheless, the adhesion to cultured IDE8 tickcells of recombinant E. coli expressing the mutant protein was abolishedwhen compared to the wild type MSP1a (Table 2). To demonstrate that theMSP1a repeats were not only necessary but sufficient to confer adhesionof recombinant E. coli to tick cells, we then constructed a chimericprotein containing the MSP1a tandem repeated peptides fused to theCOOH-terminus of the MSP1b. MSP1b did not confer an adhesive phenotypewhen expressed in E. coli (Table 3). However, E. coli expressing thechimeric protein adhered to cultured IDE8 tick cells at levelscomparable to the wild type MSP1a-expressing E. coli (Table 3).

[0053] The capacity of MSP1a to hemagglutinate bovine erythrocytes wasalso mediated by the tandem repeats. Recombinant E. coli expressing theMSP1a lacking the tandem repeats were unable to hemagglutinate bovineerythrocytes (Table 2) while the chimeric MSP1b>MSP1a-repeats proteinexpressed in E. coli conferred to recombinant bacteria a higherhemagglutination capacity (Table 3) when compared to wild type MSPs.TABLE 2 Hemagglutination of bovine erythrocytes and adhesion to culturedtick IDE8 cells by recombinant E. coli expressing A. marginale (Oklahomaisolate) MSP1a wild type and mutant protein without repeats Plasmidcarried by recombinant E. coli No pFLC1a pAF0R1 p33 plasmid Relevantprotein expressed MSP1a MSP1a-no None None repeats mutant No. of CFU(mean ± SD) 500 ± 141 14 ± 18 231 ± 129 0 recovered from IDE8 cells (N =3) Average fold increase over 2 — — — p33 control P (Student's t-Test)0.05 — — — Average fold decrease over — 36 — — MSP1a (OK) P (Student'st-Test) —  0.02 — — Hemagglutination of 1  0 0 0 bovine erythrocytes (N= 3)^(a)

[0054] TABLE 3 Hemagglutination of bovine erythrocytes and adhesion tocultured IDE8 tick cells of E. coli expressing wild type MSP1a or MSP1b(Oklahoma isolate) and MSP1b > MSP1a-repeats mutant proteins Plasmidcarried by recombinant E. coli pFLC1a pFLC1b2 pF1bRNO4 Relevant proteinexpressed MSP1a MSP1b MSP1b > MSP1a- repeats No. of CFU recovered from975 ± 742 18 ± 17 530 ± 325 IDE8 cells (Ave ± SD) (N = 2) Average foldincrease over 54 — 29 pFLC1b2 (MSP1b) Hemagglutination of bovine  1 4  5erythrocytes (N = 2)^(a)

[0055] Accordingly, it can be appreciated that subunits derived fromMSP1a are useful as well in the inventive vaccine compositions. Theinclusion of MSP1a region(s) effecting MSP1a biological function couldenhance the host immune response directed against relevantimmunoprotective epitopes.

[0056] The preparation of vaccines utilizing as distinct antigeniccomponents MSP1a is easily accomplished using well known methods andtechniques. The vaccine and/or antigen preparation is combined into aformulation in an amount effective to provide for a protective immuneresponse against infection with A. marginale. A protective immuneresponse against A. marginale decreases the clinical signs ofanaplasmosis. Clinical symptoms of anaplasmosis include a reduction inpacked red cell volume of about 25 to 80% and parasitemia of the redblood cells of about 15 to 70%. A decrease in the symptoms ofanaplasmosis includes prevention of the reduction in the packed red cellvolume and a decrease in percent parasitemia. Preferably, a protectiveresponse includes packed red cell volume change of 25% or less comparedwith control animals and/or a decrease in parasitemia to about 5 to 25%of the red blood cells or less depending on the conditions. Measurementsof packed red cell volume and percent parasitemia are conducted usingstandard methods. Vaccine preparations are combined with physiologicallyacceptable carriers to form vaccines. The preferred physiologicallyacceptable carrier is an oil-based adjuvant.

[0057] Preferably, the inventive vaccine formulation is set to containabout 100 micrograms of recombinant antigens associated to E. colimembranes in an oil-based adjuvant such as Xtend® III (GrandLaboratories, Larchwood, Iowa).

[0058] The vaccines may be administered by a variety of routes includingintravenously, intraperitoneally, intramuscularly, and subcutaneously.The preferred route of administration is subcutaneous. The vaccine canbe administered in a single dose or multiple doses until a protectiveeffect is achieved.

[0059] While the invention has been described with a certain degree ofparticularity, it is understood that the invention is not limited to theembodiment(s) set for herein for purposes of exemplification, but is tobe limited only by the scope of the attached claim or claims, includingthe full range of equivalency to which each element thereof is entitled.

BIBLIOGRAPHY

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What is claimed is:
 1. A vaccine composition for inducing an immuneresponse in a ruminant, said vaccine composition comprising at least oneantigen member of the group comprising (i) recombinant MSP1a surfaceprotein antigen of A. marginale, (ii) a subunit of recombinant MSP1asurface protein antigen of A. marginale and (iii) recombinant MSP1asurface protein antigen or subunits thereof in combination with antigenpreparation derived from A. marginale infected cultured tick IDE8 cells,wherein said vaccine composition further comprises a pharmaceuticallyacceptable carrier or diluent.
 2. The vaccine according to claim 1wherein said antigen member is recombinant MSP1a surface protein antigenof A. marginale.
 3. The vaccine according to claim 1 wherein saidantigen member is a subunit of recombinant MSP1a surface protein antigenof A. marginale.
 4. The vaccine according to claim 1 wherein saidantigen member is recombinant MSP1a surface protein antigen or subunitsthereof in combination with antigen preparation derived from A.marginale infected cultured tick IDE8 cells.
 5. A method for inducing animmune response in a ruminate to provide immune protection which reducesthe severity of or infection by A. marginale, said method comprisingadministering to said ruminant an effective amount of the vaccinecomposition of claim
 1. 6. The method according to claim 5, wherein saidantigen member is recombinant MSP1a surface protein antigen of A.marginale.
 7. The method according to claim 5 wherein said antigenmember is a subunit of recombinant MSP1a surface protein antigen of A.marginale.
 8. The method according to claim 5 wherein said antigenmember is recombinant MSP1a surface protein antigen or subunits thereofin combination with antigen preparation derived from A. marginaleinfected cultured tick IDE8 cells.